Apoptosis-related compounds and their use

ABSTRACT

Apoptosis-related antigenic compounds comprising an exposed antigenic site having the amino acid sequence  
                             SEQ ID NO: 1:         Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp         1               5                    10                
 
     or a functionally equivalent sequence comprising at least the sequence Ala Leu Asp are disclosed. Further, several applications and uses based on such compounds are included, such as use of the compounds in medicaments, nucleic acid sequences encoding the amino-acid sequence of such a peptide compound or protein fragment comprising the C-terminal amino acid sequence Ala Leu Asp, anti-sense nucleic acid sequence, vector comprising such a nucleic acid sequence, antibody or antigen-binding peptide recognizing such an antigenic compound and use thereof in a medicament, agents regulating the liberation of protein fragments comprising the amino-acid sequence SEQ ID NO: 1 or functionally equivalent sequences comprising the C-terminal amino acid sequence Ala Leu Asp, a method of determining the occurrence of cell apoptosis in a sample of biological material, especially in the diagnosis of degenerative diseases and cancer, or monitoring of the effect of therapy, a method of treating diseases with involvement of apoptosis, such as cancer and degenerative diseases, a method of creating cancer cells, and as carrier for prophylactic, therapeutic or diagnostic use.

[0001] The present invention relates to apoptosis-related antigeniccompounds, such as fragments of cytokeratin 18, and antibodies orantigen-binding peptides recognizing said compounds, which are useful inmedicaments, diagnostics and methods for the detection, monitoring,measurement and regulation of the type of cell death called programmedcell death or apoptosis. The determination of the occurrence of cellapoptosis may further be used for the monitoring of the effect oftherapeutic treatment.

BACKGROUND OF THE INVENTION

[0002] Apoptosis is seen in all sorts of higher eucaryocytes from plantsand insects to vertebrates (Kroemer G, Petit P, Zanzami N, VayssiereJ-L, Mignotte B: The biochemistry of programmed cell death. The FASEBJournal 9:1277-1287 (1995)). Apoptosis is a general phenomenon of vitalimportance. Decreased apoptosis leads to malformation, cancer andautoimmune disease, and enhanced apoptosis results in degenerativediseases, acute diseases such as infection by toxin-producingmicroorganisms and in rejection of transplanted organs.

[0003] Therefore, the detection, monitoring, measurement and regulationof apoptosis are important factors in the diagnosis and therapeutictreatment of the mentioned conditions.

[0004] Recently, Caulin C., et al (The Journal of Cell Biology, Vol 138,pp. 1379-1394) studied caspase cleavage of Keratin 18 and reorganizationof intermediate filaments during epitelial cell apoptosis. Keratin 18was cleaved in vitro by caspase −6, −3, and −7, and it was stated thatthe cleavage site common for the three caspases was the sequence VEVD/A,located in the conserved L1-2 linker region of K18.

DESCRIPTION OF THE INVENTION

[0005] During research work aiming at finding new useful specificmonoclonal antibodies reacting with human cytokeratin 18 (CK 18), inaddition to those already at hand, it was surprisingly found that amonoclonal antibody (MAb) (obtained after immunization of mice with aspecified part of the amino-acid sequence of CK 18) recognized earlyapoptotic changes in cultured epithelial cells. This MAb was designatedM30.

[0006] A detailed analysis, including synthesis and assay of a largenumber of amino-acid (aa) sequences revealed, that the specific epitopeand binding-site for the MAb M30 consists of the aa sequence:EDFNLGDALD. This 10 aa peptide sequence starts from the second coil ofhuman CK 18 and represents aa No.383-392. This specific epitope is aneo-epitope liberated during apoptisis and it is not exposed in theintact CK 18 molecule. The complete aa sequence of CK 18 was publishedby Oshima et al. (Oshima R. G., Millian J. L., and Ceccena G. (1986).Comparison of mouse and human keratin 18: a component of intermediatefilaments expressed prior to implantation. Differentiation 33:61-68.).

[0007] The different aspects of the present invention are based on theoptimal binding site of the mAb M 30, i.e. the amino-acid sequenceEDFNLGDALD, SEQ ID NO: 1: SEQ ID NO: 1: Glu Asp Phe Asn Leu Gly Asp AlaLeu Asp  1               5                   10

[0008] or functionally equivalent sequences comprising the sequence AlaLeu Asp.

[0009] Initially it was believed that an important part for apoptosis isrelated to cleavage by caspase enzymes immediately after DALD (Asp AlaLeu Asp), since this sequence occures in CK18 and caspases are known tocleave after DXXD wherein X stands for not defined aa.

[0010] However, the sequence DALD does not occur in such otherintermediate filaments as CK 1-8, vimentin, desmin, different actins,neurofilaments, and lamins. In these the following sequences are found:LALD, MALD, MALD, VALD, MALD, AND LALD, respectively.

[0011] Since the mAb M 30 recognizes apoptotic changes in non-epithelialcells such as cardiac muscle, mega-karyocytes, myeloblasts and neuraltissues of fetus—which are not known to contain CK1 8—it is now believedthat detection of protein fragments having the C-terminal sequence ALDindicates early apoptosis.

[0012] Thus, one aspect of the invention is directed to anapoptosis-related antigenic compound which specifically binds to anantibody which in turn specifically binds to the amino-acid sequence SEQID NO: 1: SEQ ID NO: 1: Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp 1               5                  10,

[0013] i.e. an apoptosis-related antigenic compound comprising anexposed antigenic site having the amino acid sequence SEQ ID NO: 1 or afunctionally equivalent sequence. Further, the functionally equivalentsequence should comprise at least the sequence Ala Leu Asp.

[0014] It is the three-dimensional structure of the exposed antigenicsite, or antibody-binding site, that defines the structure of thebinding portion of an antigenic compound which specifically binds to thecorresponding three-dimensional structure of an antibody. The antigeniccompound of the invention therefore comprises compounds which are basedon the structure of the SEQ ID NO:1, i.e. the sequence as such or afunctionally equivalent sequence, i.e. homologous in function,comprising at least the sequence Ala Leu Asp, such as compounds havingreplacements of one or several amino acids in the above specifiedamino-acid sequence with other molecular parts, D- forms of the aminoacids, non-natural amino acids and/or derivatives, as long as thethree-dimensional structure of the SEQ ID NO:1 is mimicked. Thereforethe common feature of the antigenic compounds of the invention is thatthey bind specifically to an antibody which in turn binds specificallyto the aa sequence SEQ ID NO:1, i.e. antigenic compounds comprising anantigenic site having the amino acid sequence SEQ ID NO: 1 or afunctionally equivalent sequence comprising at least the sequence AlaLeu Asp.

[0015] It can be mentioned that intact CK 18 molecules have theabove-specified sequence hidden, and can therefore not react withantibodies specifically binding to said sequence, thereby making therecognition specific to apoptosis, i.e. those cases where the CK 18molecule has been already cleaved after the C-terminal Asp of thesequence Asp Ala Leu Asp.

[0016] In a preferred embodiment of this aspect of the invention, theapoptosis-related compound is a peptide or a protein fragment comprisingthe C-terminal amino acid sequence Ala Leu Asp. The peptide may be ahomologue to, an extension of or a truncation of the SEQ ID NO: 1, i.e.may have a homologous sequence having some amino-acid substitutions,extensions, truncations and/or deletions which do not lead to theelimination of the capability of the peptide to bind to the sameantibodies as the amino-acid sequence SEQ ID NO: 1.

[0017] In another preferred embodiment of this aspect of the inventionthe antigenic compound of the invention is a peptide or a proteinfragment comprising the C-termina amino acid sequence Xaa Ala Leu Asp,wherein Xaa is selected from Asp, Leu, Met, and Val.

[0018] An example of a peptide according to the invention is anoligopeptide having the amino-acid sequence

[0019] SEQ ID NO: 1: SEQ ID NO: 1: Glu Asp Phe Asn Leu Gly Asp Ala LeuAsp  1               5                  10.

[0020] Another example is an oligopeptide having the amino-acid sequence(EDGEDFNLGDALDSSNSMQT)(i.e. SEQ ID NO: 1 extended by 3 amino acids atthe N-terminal and 7 at the C-terminal)

[0021] SEQ ID NO: 2: SEQ ID NO: 2: Glu Asp Gly Glu Asp Phe Asn Leu GlyAsp Ala Leu Asp Ser Ser Asn 1               5                   10                  15 Ser Met GlnThr              20     .

[0022] Yet another example is an oligopeptide having the amino-acidsequence (LEDGEDFNLGDALDSSNS)

[0023] SEQ ID NO: 3: SEQ ID NO: 3: Leu Glu Asp Gly Glu Asp Phe Asn LeuGly Asp Ala Leu Asp Ser Ser 1               5                   10                  15 Asn Ser.

[0024] These peptides having the amino acid sequences SEQ ID NO: 2 and 3have been synthesized, and in an ELISA they bind to the mAb M 30[relative activity: 100 for SEQ ID NO: 1; 11 for SEQ ID NO: 2; and 8.3for SEQ ID NO: 3, respectively]. However, these two peptides are notexpected to represent cleaved fragments of CK18 specific for apoptosis,since the mAb M 30 cannot bind to CK18 fragments until they expose theneo-epitope having the C-terminal sequence Ala Leu Asp.

[0025] Still another example is an oligopeptide having the amino-acidsequence (GEDFNLGDALD)

[0026] SEQ ID NO: 4: SEQ ID NO: 3: Gly Glu Asp Phe Asn Leu Gly Asp AlaLeu Asp  1               5                  10

[0027] In the present specification and claims, specific binding of anantigenic compound to an antibody, or specific binding of an antibody toan amino-acid sequence, requires an affinity constant of at least 10⁷liters/mole, preferably at least 10⁹ liters/mole.

[0028] In an embodiment of this aspect of the invention the antigeniccompound is coupled to a carrier and/or label.

[0029] The carrier may be e.g. plastic surfaces, such as microplates,beads etc.; organic molecules such as biotin; proteins, such as bovineserum albumin; peptide linkers, polypeptides e.g. resulting in fusionproteins.

[0030] The label may be selected from a variety of different types oflabels, such as radioactive isotopes, enzymes, fluorescent markers, etc.

[0031] Another aspect of the invention is directed to an antigeniccompound according to the invention for use in a medicament.

[0032] In a preferred embodiment of this aspect of the invention anantigenic compound according to the invention is used in the productionof a medicament for inhibition of ceil apoptosis.

[0033] Inhibition of cell apoptosis may be desirable in the treatment ofdegenerative conditions, such as anorexia, AIDS, transplantation oforgans, psoriasis, and Alzheimer's disease.

[0034] Yet another aspect of the invention is directed to an isolated orrecombinant nucleic acid sequence comprising a nucleotide sequence whichencodes an amino-acid sequence of a peptide or protein fragmentaccording to the invention.

[0035] A nucleic acid sequence of the present invention may be either inthe form of RNA or DNA (including cDNA, genomic DNA and synthetic DNA).The DNA may be double-stranded or single-stranded. When the DNA sequenceis single-stranded, it may be either the coding sequence (sense strand)or non-coding sequence (anti-sense strand). The nucleic acid sequencesof the invention can be designed from the amino-acid sequence of apeptide or protein fragment comprised by the invention with the aid ofthe genetic code.

[0036] The nucleic acid sequences of the invention can be used in theproduction of proteins, polypeptides , oligopeptides and peptides of theinvention. In a particular embodiment of this aspect of the invention,the nucleic acid sequence is an anti-sense sequence based on the nucleicacid sequence of the invention, which is complementary, in whole or atleast a part of the SEQ ID NO: 1 encoding part, to a sense sequenceencoding a peptide or a protein fragment of the invention. Whenintroduced into a cell, the anti-sense nucleic acid sequence can inhibitthe expression of the gene encoded by the sense strand.

[0037] This aspect of the invention also involves a vector comprising anucleic acid sequence of the invention. Such a vector may be used in theproduction of a peptide or protein fragment of the invention or possiblyin the regulation of the amount of protein fragments comprising theamino acid sequence SEQ ID NO: 1 or functional equivalents thereofcomprising at least the sequence Ala Leu Asp in target cells, in vivo orin vitro. In a preferred embodiment of the invention, the vector is aplasmid.

[0038] Still another aspect of the invention is directed to an antibodyor antigen-binding peptide recognizing an antigenic compound accordingto the invention.

[0039] The antibody of the invention may be a monoclonal antibody ormonospecific polyclonal antibody recognizing or specifically binding toan antigenic compound according to the invention, i.e. recognizing orspecifically binding to the amino-acid sequence SEQ ID NO: 1 or afunctionally equivalent sequence comprising at least the sequence AlaLeu Asp. The antibody of the invention can be prepared by usingimmunization procedures well known in the art, or by well known methodsbased on recombinant technology making use of suitable host cells ofeukaryotic or prokaryotic origin.

[0040] The antigen-binding peptide recognizing an antigenic compoundaccording to the invention may be the antigen-binding part of anantibody, or a protein or peptide which has an amino-acid sequence thatat least in the part corresponds to the amino-acid sequence SEQ ID NO: 1or its functional equivalent comprising at least the sequence Ala LeuAsp but has amino acids with the opposite charges. Such anantigen-binding peptide will function as an antagonist.

[0041] In the development of antagonists the following guideline may beused:

[0042] ∘=hydrophobic amino acid

[0043] +=positive amino acid

[0044] −=negative amino acid

[0045] ±=neutral amino acid SEQ ID NO: 1 :      E D F N L G D A L D     − − o ± o ± − o o − corresponding site      + + o ± o ± + o o + ofantagonist : e.g. K R W Q F G R L A R

[0046] A number of antagonist candidates may be designed with the aid ofthe above guideline, especially variants where K and R are used for the+positions.

[0047] In an embodiment of this aspect of the invention the antibody orantigen-binding peptide is coupled to a carrier and/or label.

[0048] The carrier may be e.g. plastic surfaces, such as microplates,beads etc.; organic molecules such as biotin; proteins, such as bovineserum albumin; peptide linkers, polypeptides e.g. resulting in fusionproteins.

[0049] The label may be selected from a variety of different types oflabels, such as radioactive isotopes, enzymes, fluorescent markers, etc.

[0050] A further aspect of the invention is directed to the antibody andantigen-binding peptide of the invention for use in a medicament.

[0051] In a preferred embodiment of this aspect of the invention anantibody or antigen-binding peptide of the invention is used in theproduction of a medicament for the stimulation of apoptosis,particularly in conditions such as malformation, cancer and autoimmunedisease, i.e. conditions relating to uncontrolled or excessive cellproliferation.

[0052] In another embodiment of the invention the produced medicament isdesirable in the treatment of diseases with involvement of enhancedapoptosis, such as many degenerative diseases, or acute diseases such asinfection by toxin-producing microorganisms or ischemic-reperfusiondamage and rejection of transplanted tissue or organs. Here, medicalcontrol of apoptosis may be of utmost importance.

[0053] Yet another aspect of the invention is directed to agentsregulating the liberation in biological material, including themammalian body or cell culture, of protein fragments comprising theC-terminal amino-acid sequence Ala Leu Asp.

[0054] Examples of such agents of the invention are nucleic acidsequences of the invention, either inhibiting (sense strand) orstimulating (anti-sense strand), the expression of proteins comprisingthe sequence SEQ ID NO: 1 or sequences specifically binding to the sameantibodies or antigen-binding peptides as the SEQ ID NO: 1, i.e.functionally equivalent sequences comprising at least the sequence AlaLeu Asp, and enzymes, enzyme activators and inhibitors.

[0055] Still another aspect of the invention is directed to a method ofdetermining the occurrence of cell apoptosis in a biological sampleincluding a sample of an organ, tissue or body fluid from a mammal,including man, wherein the presence of protein fragments comprising theC-terminal amino-acid sequence Ala Leu Asp, is determined.

[0056] In a preferred embodiment of this aspect of the invention thedetermination is performed with an immunological assay using theantibody of the invention.

[0057] In another preferred embodiment of this method the rate ofoccurrence of cell apoptosis is determined. The determined rate of cellapoptosis may be used in the diagnosis of diseases with involvement ofapoptosis, such as degenerative diseases and cancer, and in themonitoring of the effect of therapy.

[0058] As origin of samples in this aspect the following may bementioned: in vivo or in vitro, any organs, normal or changed, tissues,specimens and fluids of the human or animal body, for example liver,lung, kidney, heart, spleen, brain, viscera, lymphatic organs,bone-marrow, reproductive organs, skeleton, muscle, skin, sensoryorgans, glands, blood, serum, urine, ascitic fluid, pleural fluid,cerebrospinal fluid, amniotic fluid, abscess fluid, wash fluids,punctures, slices and any preparation of cellular or fluid origin inthis context.

[0059] The determination of said protein fragments may be performed byany technique which can detect and preferably quantify the amount ofpeptide fragments comprising the C-terminal amino acid sequence Ala LeuAsp in a sample of body fluid. Preferably the protein fragments compriseat least the amino acid sequence SEQ ID NO: 1 or sequences specificallybinding to the same antibodies or antigen-binding peptides as the SEQ IDNO: 1, i.e. functionally equivalent sequences comprising at least thesequence Ala Leu Asp. Monoclonal antibodies which specifically bind tosaid fragments can be used in different immunoassays, optionally labeledin accordance with the actual assay used.

[0060] In one embodiment of said method of the invention, thedetermination is performed with the aid of an immunoassay. Also,detection systems adapted for the use of antibody-site carryingstructures and/or antibodies may be used. Some examples of the numerousimmunoassays which may be used in the invention are Enzyme-linkedimmunosorbent assay (ELISA),

[0061] Radioimmunoassay (RIA, IRMA), Fluorescence immunoassay (FIA),Chemiluminescent enzyme-labeled immunometric assay, Luminescenceimmunoassay (LIA), Dissociation enhancement time-resolvedfluoroimmunoassay (DELFIA). The assay may be manual or automatic.

[0062] Further, an aspect of the invention is directed to a method oftreating diseases with involvement of apoptosis, such as cancer anddegenerative diseases, in a patient comprising administration of a cellapoptosis-regulating amount of an antibody or antigen-binding peptideaccording to the invention or of an antigenic compound according to theinvention, to said patient.

[0063] Another aspect of the invention is directed to a method ofcreating cancer cells comprising administration of a cellapoptosis-inhibiting amount of an antigenic compound according to theinvention to a cell culture or a laboratory animal. Such a cell cultureor laboratory animal can be used in the production of desiredpolypeptides or proteins or in the evaluation of candidate anti-cancerdrugs.

[0064] An additional aspect of the invention is directed to a carrierfor prophylactic, therapeutic or diagnostic use comprising an antibodyor antigen-binding peptide of the invention. Such a carrier of theinvention will function as a targeting substance finding fragmentscomprising the C-terminal amino acid sequence Ala Leu Asp, such as SEQID NO: 1 or functionally equivalent sequences comprising at least thesequence Ala Leu Asp, and it may be coupled to a variety of medicamentsand/or labels for prophylactic, therapeutic or diagnostic purposes.

[0065] The present invention will now be further illustrated byreference to the following description of experiments and specificembodiments of the invention, which are not to be considered aslimitations to the scope of the invention defined in the claims.

[0066] Synthesis of Peptides of the Invention

[0067] The peptides of the invention can be produced by any known methodof producing an amino-acid sequence, such as, controlled degradation ofa purified protein by proteases or other chemical methods (Allen G.,Sequencing of proteins and peptides, 1989, Elsevier Science PublishersB.V.). Chemical synthesis is commonly performed by coupling of the aminoacid residues or peptide fragments to one another in correct order inliquid phase to produce the desired peptide. Another common strategy isthe coupling of the amino acids to one another starting with a solidphase (resin) to which the C-terminal of the last amino acid of thesequence is coupled, whereupon the C-terminal of the penultimate aminoacid is coupled to the N-terminal of the last amino acid, etc., finallyreleasing the built-up peptide from the solid phase (so calledsolid-phase technique).

[0068] The oligopeptides made in order to illustrate the invention weresynthesized using the multipin peptide synthesis approach usingpolyethylene supports derivatized with an acid handle (Cf. Valerio, R.M., Bray, A. M. and Maeji, N. J. (1994) Multiple peptide synthesis onacid-labile handle derivatized polyethylene supports. Int. J. PeptideProtein Res. 44:158-165.). The synthesis was carried out on detachablepins grafted with hydroxyethylmethacrylate and functionalized with thetrifluoroacetic acid-labile Rink amide forming handle. Peptidesrepresenting both the N and C terminal truncation series of the sequenceEDFNLGDALD and other sequences were synthesized. Peptides were cappedwith biotin using the tetra peptide linker sequence—SGSG- or -SGSB-(B=βalanine), and attached to streptavidin coated plates in theenzyme-linked immunosorbent assay.

[0069] The aa sequences were checked for purity by reverse phase highperformance liquid chromatography (RP-HPLC) and by ion spray massspectrometry (IS-MS) (Cf. Van Dorsselear et al., (1990) Application ofelectrospray mass spectrometry to characterization of recombinantproteins up to 44 kDa. Biomed.Environ.Mass Spectrom. 19:692-704).

[0070] Ion spectra were collected as positive ions, obtained byvaporizing the peptide from acidic (trifluoroacetic or acetic acid)solution. Percentage of total ion count versus molecular weight (Dalton)was derived from the spectrum of raw mass-to-charge ratio versus ioncount. The IS-MS purity value is the ion count, attributable to thetarget peptide, expressed as percentage of the total ion count.

[0071] Immunization of Mice

[0072] Preparation of Immunizing Material

[0073] The starting material for antigen purification consisted of thesupematant of cell culture medium from human colon carcinoma cell lineWIDR (ATCC No. CCL 218) (Rydlander L, Ziegler E, Bergman T, Schöberl E,Steiner G, Bergman A-C, Zetterberg A, Marberger M, Björklund P, Skern T,Einarsson R and Jörnvall H (1996): Molecular characterization of atissue-polypeptide-specific-antigen epitope and its relationship tohuman cytokeratin 18. Eur.J.Biochem. 241:309-314.) The first steputilized precipitation with 50% (mass/vol) ammonium sulfate. This wasfollowed by hydrophobic-interaction chromatography on phenyl Sepharosein 14 mM phosphate/85 mM ammonium sulfate, pH 7.5. After washing of thecolumn, hydrophobic proteins were eluted with water, and fractionscollected. The third step consisted of Sephacryl S-300 exclusionchromatography in 8 M urea, 0.1 M Tris/HCI, pH 8.0. In the fourth step,Q Sepharose, equilibrated with 8 M urea in 0.1 M Tris/HCL, pH 8.0. wasemployed.

[0074] The fraction eluted at 0.12 M NaCI was about 882 times purifiedand contained two active components, one of 13 kD, another of 22 kD.They were identified as subtypes of cytokeratin 18, both ending at aa396, i.e. fragments comprising a C-terminal aa sequence ALD.

[0075] Immunization Procedure

[0076] Four BalbC mice received one intraperitoneal (i.p.) injection of100 μl of Freund's complete adjuvant suspended in 100 μl balanced saltsolution (PBS) containing 107 μg of the first batch of the purifiedantigen. Three, six and nine weeks later, the mice received similarinjections and amounts of an additional batch of the antigen, but withincomplete instead of complete Freund's adjuvant. Eight weeks after thelast injection, a booster injection of 53 μg of a new batch of antigenwithout adjuvant in 100 μl PBS was given i.v. and the same amount wasintroduced i.p.

[0077] Immunization of BalbC mice with an appropriate dose of thesynthesized peptide EDFNLGDALD, coupled to the lipopeptide adjuvant:(Pam3cysSer(Lys)4, Boehringer 1428 764,lot 1312 7024), will result indevelopment of immuno-competent cells producing monoclonal antibodiesagainst the synthetic oligopeptide.

[0078] Hybridization

[0079] Three days after the booster injection of the mice, the spleencells were hybridized with mouse myeloma cells (cell line: P3×63-Ag8.653, G.Köhler)). Mediums from a large number of resulting hybridomacultures were reacted with rabbit anti-mouse antibodies (solid phase),whereupon immunizing antigen and blocking mouse globulin were added,followed by HRP-labeled, detecting antibody against CK 18. Byimmunocytochemical analyses it was observed that one of the resultinghybrid cell lines (No. 30) produced antibodies that reacted withcultured cells, that were neither vital nor necrotic. Among the cellstested were HeLa cells (Gey 1952), bladder carcinoma cell line T24,mammary cell line T47d, colon carcinoma cell line WiDr CCL 218 grown inIMDM 90%+10% fetal bovine serum+2 mm L-glutamine and 10 μg Gentamycine.The unexpected observations gave rise to detailed studies of theantibody specificity by immunochemical and immunocytochemicaltechniques. It became evident that the MAb, designated M30, reactedspecifically with apoptotic cells and fluids.

[0080] Immunochemistry

[0081] In order to characterize the MAb M30-epitope chemically, the M30was tested against a large number of synthetic peptides, that wereespecially made for this purpose. It was found that the optimal specificepitope consisted of the aa sequence EDFNLGDALD. This and relatedsequences are presented in Table 1 which shows the reactivity ofamino-acid sequences with the monoclonal antibody M 30 of the invention.TABLE 1 Absorbance Peptide No. Link to Biotin at 405/492 nm Sequence 63-SGSG- 0.05 LEDGEDFNLGDAL 61 -SGSG- 0.1 LEDGEDFNLGDALDS 46 -SGSG- 0.77DGEDFNLGDALD 45 -SGSG- 1.05 EDGEDFNLGDALD 47 -SGSG- 1.23 GEDFNLGDALD 302-SGSB- 1.41 GEDFNLGDALD 301 -SGSB- 1.46 EDFNLGDALD 401 -SGSB- 0.62DFNLGDALD 402 -SGSB- 0.27 FNLGDALD 403 -SGSB- 0.14 NLGDALD 404 -SGSB-0.1 LGDALD 405 -SGSB- 0.1 GDALD 406 -SGSB- 0.1 DALD

[0082] As is evident from Table 1, gradual shortening of the sequencefrom the amino end to EDFNLGDALD, gave maximal activity. At the otherend, removal of the last D (Asp) or addition of an S (Ser) to the last Dresulted in complete loss of activity.

[0083] The need for the last D in DALD is clear-cut and should be seenin connection with what is known about structures required forapoptosis: Active caspase-3 enzyme recognizes an DXXD pattern in targetsubstrates (Nicholson D. W., Ali A, Thornberry N. A., Vaillancourt J.P., Ding C. K., Gallant M, Gareau Y, Griffin P. R., Labelle M, LazebnikY. A., Munday N. A., Raju S. R., Smulson M. E., Yamin T-T, Yu V. L.,Miller D. K. , et al. (1995). Identification and inhibition of theICE/CED-3 protease necessary for mammalian apoptosis. Nature.376:37-43.). In addition, both caspase-6 and caspase-7 are able tocleave substrates with a DXXD sequence (Talanian R. V., Quinlan C,Trautz S, Hacket M. C., Mankovich J. A., Banach D, Ghayur T, Brady K. D.Wong W. W. (1997). Substrate specificities of caspase family proteases.J.Biol.Chem.272:9677-9682.). So far, the new sequence described here hasnot been known before to indicate apoptosis.

[0084] That proteases of the caspase subfamily are involved in mammalianapoptosis is supported by compelling evidence ranging from observationof DXXD-X cleaving activity and the activation of proenzymes inapoptotic cells, and the inhibition of apoptosis in a variety of systemsby specific substrate-mimicking peptide inhibitors (Nobel S.(1997).Thiol redox state in apoptosis: Physiological and toxicant modulation.Dissertation, Karolinska Institutet, Stockholm, Sweden. ISBN91-628-2502-X.).

[0085] Immunocytochemistry

[0086] Today there are several methods for the detection of apoptosis,but none is based on a defined reacting part of cytokeratin 18 withknown amino acid sequence. The most widely used methods to identifyapoptotic cells are light and electron microscopic analysis, flowcytometry, agarose gel electrophoresis, in situ nick-end labeling (ISEL)and the TdT-mediated dUTP Nick End Labeling (TUNEL)-technique.

[0087] To substantiate the preliminary findings, and to fit theexpression of the epitope, recognized by the MAb M30, the effect of thedifferent apoptosis systems were analyzed and a comparison made betweenM30-immunocytochemistry and accepted apoptosis assays. Comparisons weremade using e.g. the non small cell lung cancer cell line MR65 with theannexin-, the TUNEL- and flow cytometric assays next to establishedmorphological criteria. Confocal laser microscopic analysis revealedthat apoptotic cells showed a bright immunofluorescence cytoplasmicstaining after incubation with the MAb M30, while viable and necroticcells turned out to be negative.

[0088] The expression of the M30-epitope correlated very well with otheraccepted staining methods for the detection of apoptotic cells i.e.annexin V-, TdT-mediated dUTP Nick End Labeling (TUNEL)-assay andmorphological criteria. The majority of the M30-positive cells were inthe “apoptotic” sub G1-peak. The expression of the M30-epitope occurredearly in the apoptotic cascade, before annexin V reactivity or positiveNick end labeling. The epitope was not detectable in vital epithelialcells. The epitope, consisting of the aa sequence EDFNLGDALD orfunctionally equivalent sequences comprising at least the sequence AlaLeu Asp, allows quantification of apoptotic cells at the level of thecytoskeleton using simple immunocytochemical techniques.

[0089] The major drawbacks of the hitherto accepted methods fordetection of apoptosis are that they are time consuming, relativelyexpensive, labor-intensive and not always specific for apoptotic cells.In addition, the TUNEL assay and the ISEL can only detect intermediateand late apoptosis.

[0090] When the effects of different apoptosis induction systems wereanalyzed and a comparison made between M30-immunocytochemistry andaccepted apoptosis-assays, it turned out, that M30 is superior toexisting apoptosis detection assays in cell lines and routinely obtainedbiopsies of human origin. M30 specifically recognizes apoptotic cells inthe early stages of the process.

[0091] Since M30 recognizes the intrinsic early marker of apoptosis,i.e. EDFNLGDALD or functionally equivalent sequences comprising at leastthe sequence ALD, it is applicable to fresh and also formalin fixed,paraffin embedded tissue sections of routinely obtained biopsies. Thisis one of the advantages of M30. Since M30-immunoreactivity alsoprecedes both loss of membrane asymmetry, as is detected by annexinV-binding and DNA fragmentation, which is detected by the TUNEL assay,it offers distinctive advantages over presently used routine assays.Since apoptosis is implicated in many diseases, the specific, earlydetection and quantification of apoptotic cells is of utmost importance.

[0092] Body Fluids Reflecting Apoptosis

[0093] When apoptotic products containing EDFNLGDALD, or functionallyequivalent sequences comprising at least the sequence ALD, are releasedfrom apoptotic cellular areas in the body into body fluids such asserum, ascites, pleura, etc., immunoreactive assays can be used with MAbM30 just as with other serum assays to detect and quantify theconcentration of special products in serum and body fluids. In this wayone can differentiate between illnesses caused by or connected withenhanced or insufficient apoptosis i.e. increased or decreased levels ofthe MAb M30 binding site EDFNLGDALD or functionally equivalent sequencescomprising at least the sequence ALD. Specimens from implicated organsor tissues can contribute to a more specific diagnosis.

[0094] Diagnostic Use of the SEQ ID NO: 1 or Functionally EquivalentSequences

[0095] The process of apoptosis can be subdivided into three phases: theinduction phase, the effector phase and the degradation phase.Cytokeratin 18 degradation and production of fragments containingEDFNLGDALD takes place very early and long before any of the knownmethods for assay of apoptosis turn positive. This makes it possible tointerfere very early with the production and function of EDFNLGDALD, orfunctionally equivalent sequences comprising at least the sequence ALD,with the aid of antagonistic medicals, specific antibodies toEDFNLGDALD, or functionally equivalent sequences comprising at least thesequence ALD, and also inhibitors or activators of the enzymes thatcontribute to the production of EDFNLGDALD or functionally equivalentsequences comprising at least the sequence ALD. By using the knowledgeobtained from the analysis of concentration and localization ofM30-positive specimens, it is possible to tell, if enhancement orinhibition of the process of apoptosis (programmed cell death) isdesirable and if one should try to influence this process by labeled orunlabeled M30-antibody, more labeled or unlabeled EDFNLGDALD, orfunctionally equivalent sequences comprising at least the sequence ALD,labeled or unlabeled antagonist to EDFNLGDALD, functionally equivalentsequences comprising at least the sequence ALD, such as KRWQFGRLAR orsimilar, or by inhibiting or enhancing the enzyme producing EDFNLGDALDor functionally equivalent sequences comprising at least the sequenceALD. The choice is based upon the exact determination of the apoptoticamino acid sequence EDFNLGDALD or functionally equivalent sequencescomprising at least the sequence ALD with the aid of the monoclonalantibody M30.

1 22 1 10 PRT Artificial Sequence Description of Artificial Sequenceepitope from mammalian protein 1 Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp1 5 10 2 20 PRT Artificial Sequence Description of Artificial Sequenceepitope from mammalian protein 2 Glu Asp Gly Glu Asp Phe Asn Leu Gly AspAla Leu Asp Ser Ser Asn 1 5 10 15 Ser Met Gln Thr 20 3 18 PRT ArtificialSequence Description of Artificial Sequence epitope from mammalianprotein 3 Leu Glu Asp Gly Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp SerSer 1 5 10 15 Asn Ser 4 11 PRT Artificial Sequence Description ofArtificial Sequence epitope from mammalian protein 4 Gly Glu Asp Phe AsnLeu Gly Asp Ala Leu Asp 1 5 10 5 4 PRT Artificial Sequence Descriptionof Artificial Sequence epitope from mammalian protein 5 Asp Ala Leu Asp1 6 4 PRT Artificial Sequence Description of Artificial Sequence epitopefrom mammalian protein 6 Leu Ala Leu Asp 1 7 4 PRT Artificial SequenceDescription of Artificial Sequence epitope from mammalian protein 7 MetAla Leu Asp 1 8 4 PRT Artificial Sequence Description of ArtificialSequence epitope from mammalian protein 8 Val Ala Leu Asp 1 9 4 PRTArtificial Sequence Description of Artificial Sequence epitope frommammalian protein 9 Ser Gly Ser Gly 1 10 13 PRT Artificial SequenceDescription of Artificial Sequence epitope from mammalian protein 10 LeuGlu Asp Gly Glu Asp Phe Asn Leu Gly Asp Ala Leu 1 5 10 11 15 PRTArtificial Sequence Description of Artificial Sequence epitope frommammalian protein 11 Leu Glu Asp Gly Glu Asp Phe Asn Leu Gly Asp Ala LeuAsp Ser 1 5 10 15 12 12 PRT Artificial Sequence Description ofArtificial Sequence epitope from mammalian protein 12 Asp Gly Glu AspPhe Asn Leu Gly Asp Ala Leu Asp 1 5 10 13 13 PRT Artificial SequenceDescription of Artificial Sequence epitope from mammalian protein 13 GluAsp Gly Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp 1 5 10 14 11 PRTArtificial Sequence Description of Artificial Sequence epitope frommammalian protein 14 Gly Glu Asp Phe Asn Leu Gly Asp Ala Leu Asp 1 5 1015 4 PRT Artificial Sequence UNSURE (4) x equals bAla 15 Ser Gly Ser Xaa1 16 9 PRT Artificial Sequence Description of Artificial Sequenceepitope from mammalian protein 16 Asp Phe Asn Leu Gly Asp Ala Leu Asp 15 17 8 PRT Artificial Sequence Description of Artificial Sequenceepitope from mammalian protein 17 Phe Asn Leu Gly Asp Ala Leu Asp 1 5 187 PRT Artificial Sequence Description of Artificial Sequence epitopefrom mammalian protein 18 Asn Leu Gly Asp Ala Leu Asp 1 5 19 6 PRTArtificial Sequence Description of Artificial Sequence epitope frommammalian protein 19 Leu Gly Asp Ala Leu Asp 1 5 20 5 PRT ArtificialSequence Description of Artificial Sequence epitope from mammalianprotein 20 Gly Asp Ala Leu Asp 1 5 21 10 PRT Artificial SequenceDescription of Artificial Sequence epitope from mammalian protein 21 LysArg Trp Gln Phe Gly Arg Leu Ala Arg 1 5 10 22 5 PRT Artificial SequenceDescription of Artificial Sequence epitope from mammalian protein 22 ValGlu Val Asp Ala 1 5

1. Apoptosis-related antigenic compound comprising an exposed antigenicsite having the amino acid sequence SEQ ID NO: 1: Glu Asp Phe Asn LeuGly Asp Ala Leu Asp  1               5                   10

or a functionally equivalent sequence comprising at least the sequenceAla Leu Asp.
 2. Antigenic compound according to claim 1, wherein thecompound is a peptide, or a protein fragment comprising the C-terminalamino acid sequence Ala Leu Asp.
 3. Antigenic compound according toclaim 2, wherein the compound is a peptide or a protein fragmentcomprising the C-terminal amino acid sequence Xaa Ala Leu Asp, whereinXaa is selected from Asp, Leu, Met, and Val.
 4. Antigenic compoundaccording to claim 2 or 3, wherein the peptide is an oligopeptide havingthe amino-acid sequence SEQ ID NO: 1: Glu Asp Phe Asn Leu Gly Asp AlaLeu Asp  1               5                  
 10.


5. Antigenic compound according to claim 2 or 3 , wherein the peptide isan oligopeptide having the amino-acid sequence SEQ ID NO: 4: Gly Glu AspPhe Asn Leu Gly Asp Ala Leu Asp. 1                5                  10


6. Antigenic compound according to any one of claims 1-5 coupled to acarrier and/or label.
 7. Antigenic compound according to any one ofclaims 1-6 for use in a medicament.
 8. Antigenic compound according toclaim 7 for use in the production of a medicament for inhibition of cellapoptosis.
 9. Isolated or recombinant nucleic acid sequence comprising anucleotide sequence which encodes an amino-acid sequence of a peptide orprotein fragment according to any one of claims 2-6.
 10. Anti-sensenucleic acid sequence based on a sense nucleic acid sequence accordingto claim
 9. 11. Vector comprising a nucleic acid sequence according toclaim 9 or
 10. 12. Vector according to claim 11, wherein the vector is aplasmid.
 13. Antibody or antigen-binding peptide recognizing anantigenic compound according to any one of claims 1 -
 6. 14. Antibody orantigen-binding peptide according to claim 13 coupled to a carrierand/or label.
 15. Antibody or antigen-binding peptide according to claim13 or 14 for use in a medicament.
 16. Antibody or antigen-bindingpeptide according to claim 15 for use in the production of a medicamentfor the stimulation of cell apoptosis.
 17. Agent regulating theliberation in biological material, including the mammalian body or cellculture, of protein fragments comprising the C-terminal amino-acidsequence Ala Leu Asp.
 18. Method of determining the occurrence of cellapoptosis in a biological sample including a sample of an organ, tissueor body fluid from a mammal, including man, wherein the presence ofprotein fragments comprising the C-terminal amino-acid sequence Ala LeuAsp is determined.
 19. Method according to claim 18 , wherein thedetermination is performed with an immunological assay using theantibody of claim 13 or
 14. 20. Method according to claim 18 or 19,wherein the rate of occurrence of cell apoptosis is determined. 21.Method according to claim 20, wherein the determined rate of cellapoptosis is used in the diagnosis of diseases with involvement ofapoptosis, such as degenerative diseases and cancer and/or monitoring ofthe effect of therapy.
 22. Method of treating diseases with involvementof apoptosis, such as cancer and degenerative diseases, in a patientcomprising administration of a cell apoptosis-regulating amount of anantibody or antigen-binding peptide according to any one of claims13-16, or of an antigenic compound according to any one of claims 1-6,to said patient.
 23. Method of creating cancer cells comprisingadministration of a cell apoptosis-inhibiting amount of an antigeniccompound according to any one of claims 1-6 to a cell culture or alaboratory animal.
 24. Carrier for prophylactic, therapeutic ordiagnostic use comprising an antibody or antigen-binding peptiderecognizing an antigenic compound according to any one of claims 1-6.